Cloning And Functional Analysis Of BOK In Orange-spotted Grouper Epinephelus Coioides

Epinephelus coioides belong to Perciformes,Serranidae,and subfamily grouper.Epinephelus,lives in cold and warm sea water,with fast growth,and high-survival rates.It is farmed in the coastal areas of south China and quickly becoming an economic species in South China and Southeast Asia as a whole.increased stocking density,and continual deterioration of the marine environment has accentuated viral and bacterial diseases in grouper aquaculture resulting in extreme economic losses.In recent years,some scholars separated a virus from the tissue of a sick cultured Grouper(Epinephelus coioides)named Singapore grouper iridovirus(SGIV).Reports of the devastating effects of SGIV infection can reach a mortality as high as 90%.As it stands,the immune response of grouper,especially in virus-infected condition/state,as well as the interactions between the host and the viruses is largely unknown.1.With regard to thesis,full length cDNA of Epinephelus coioides BOK(EcBOK)gene was successfully cloned by RACEPCR.The full length cDNA of EcBOK gene is 1433 bp,containning a 5' untranslated region(5'-UTR)of 236 bp,3' untranslated region(3'-UTR)of 574 bp,the full length of open reading frame(ORF)of 633 bp which coding 210 amino acids with an estimated molecular weight(MW)of 23.18 KDa and an estimated isoelectric point(p I)of 8.47.The deduced protein of Ec BOK contained a transmembrane domain(TM),four Bcl 2 homologous domains(BH1-BH4),EcBOK encoded amino acid peptide which shared high homology with Stegastes partitus(97%),Oreochromis niloticus(93%),Takifugu rubripes(90%)and Gallus gallus(69%).Phylogenetic analysis showed that EcBOK had the closest distance with Stegastes partitus.2.EcBOK was distributed in all examined tissues,with high expression in the heart and gill,and significant expression in the two immune organs,head kidney and spleen.when GS was infected by SGIV in vivo,the result showed that the relative expression of EcBOK gene was increased in hours.This implied that the Ec BOK gene may play a vital role in the process of antiviral immune response.Some studies have shown that SGIV can infect FHM cells which are dead in typical apoptosis pathway.considering this paper,EcBOK expression vectors were successfully built,EcBOK expression vectors and empty vectors were then transfected into FHM cells,respectively.3.SGIV infected FHM cells which initially were transfected this two vectors.The result demonstrated EcBOK inhibit replication of SGIV and the process of virus-induced apoptosis.In summary,EcBOK plays a vital role in Epinephelus coioides antivirus immune response,which not only help provide new theoretical basis for researching the functions of BOK,but also provides a new strategy for fish antivirus immune response mechanisms.